Pak. J. Bot., 50(3): 955-961, 2018.

TREHALOSE-INDUCED IMPROVEMENT IN GROWTH, PHOTOSYNTHETIC

CHARACTERISTICS AND LEVELS OF SOME KEY OSMOPROTECTANTS IN

SUNFLOWER (HELIANTHUS ANNUUS L.) UNDER DROUGHT STRESS

FIRDOS KOSAR1, NUDRAT AISHA AKRAM1*, MUHAMMAD ASHRAF2,

MUHAMMAD SADIQ1 AND FAHAD AL-QURAINY3

1Department of Botany, Government College University, Faisalabad, Pakistan

2Pakistan Science Foundation, Islamabad, Pakistan 3Department of Botany, King Saud University, Saudi Arabia

*Corresponding author's email: nudrataauaf@yahoo.com

Abstract

Accumulation of trehalose, a sugar rarely found in plants, is believed to be involved in improving drought stress tolerance

in different plant species. To assess the influence of trehalose on regulation of different gas exchange attributes, chlorophyll

contents and osmoprotectants of sunflower (Helianthus annuus L.) plants, an experiment was conducted under drought stress

conditions. Fifteen day-old seedlings of two sunflower cultivars i.e. Hysun 33 and FH 598 were exposed to non-stress (100%

field capacity) and water limited stress (60% field capacity). After 30 days of drought stress treatments, four levels of trehalose

(0, 10, 20 and 30 mM) were applied to the plant leaves of both sunflower cultivars. Water deficit conditions suppressed shoot

and root fresh and dry weights, chlorophyll a and b contents, photosynthetic rate (A), transpiration rate (E), stomatal

conductance (gs), sub-stomatal CO2 concentration (Ci), and Ci/Ca ratio of both sunflower cultivars while it significantly

enhanced the WUE as well as accumulation of proline and glycinebetaine (GB)contents in both sunflower cultivars. Foliar-

applied different levels of trehalose significantly improved shoot fresh and dry weights, root dry weight, chlorophyll a and b, A,

E, gs, Ci, Ci/Ca, WUE, proline and GB contents under water stress conditions. Of both sunflower cultivars, cv. Hysun 33 was

superior in shoot and root fresh and dry weights, A, E, gs, Ci, Ci/Ca under water limited conditions, whereas cv. FH 598 in

WUE. Of all trehalose levels used, 20mM and 30mMwere more effective for improving shoot fresh weight, chlorophyll b, gs,

Ci, and Ci/Ca in both sunflower cultivars. Overall, exogenous application of trehalose was effective in enhancing drought stress

tolerance in sunflower plants by improving osmoprotection and gas exchange characteristics.

Key words: Chlorophyll pigments, Gas exchange characteristics, Glycinebetaine, Proline, Trehalose.

Introduction

Various environmental stresses including drought

caused a significant decline in crop productivity every year

(Sadiq et al., 2017). Drought stress is one of the main

abiotic stresses that influence adversely crop development

and yield (Sadiq et al., 2016). Water limited regimes cause

a prominent decrease in plant photosynthetic processes,

mainly due to impaired stomatal conductance, which

reduces CO2 entry into leaf tissues or due to impairment of

the activity of Rubisco, a key enzyme of photosynthetic

metabolism (Ashraf & Harris, 2013). Water deficit

conditions also lower down considerably plant water

contents and perturb different metabolic processes

including protein synthesis, rate of photosynthesis and

respiration, synthesis of primary and secondary

metabolites, generation of reactive oxygen species (ROS),

hormonal homeostasis and levels of essential nutrients in

plants (Ahmad et al., 2010). To cope with such adverse

conditions caused by abiotic stresses, plants adopt different

mechanisms including accumulation of compatible organic

osmolytes through their metabolic pathways (Ashraf,

2009). Plants can regulate cellular osmotic adjustment, a

key physiological protective process occurring in plants

under water scarce conditions. Water-stressed plants

possess the ability to accumulate a variety of organic

osmolytes but the extent of accumulation varies from

species to species and from variety to variety. However, the

intrinsic levels of the osmolytes have been reported to be

linked to plant drought tolerance (Khan et al., 2010). On

the basis of pattern of accumulation of organic osmolytes,

plants have been categorized as high, low and non-

accumulators (Slama et al., 2015). For raising the levels of

osmolytes particularly in low accumulators or non-

accumulator’s efforts are underway to genetically engineer

such plants to enable them to produce/accumulate desired

levels of the key osmolytes. However, considering this

approach a very cost-intensive and time-consuming,

alternatively a shot-gun approach wherein osmolytes are

supplied to plants through exogenous means so as to raise

the intrinsic concentrations of osmolytes to a desired level.

A variety of organic osmolytes are being supplied

exogenously to plants for achieving enhanced drought

tolerance. They include soluble sugars, amino acids

including proline, quaternary ammonium compounds

including glycinebetaine, synthetic plant growth

regulators, etc. (Rao et al., 2012). Of different soluble

sugars known for exogenous supply to plants, trehalose

has gained ground as an effective osmolyte for enhancing

drought tolerance in most plants (Govind et al., 2016).

Trehalose, a non-reducing disaccharide, is believed to

protect biological membranes and molecules in living

organisms during water deficiency (Akram et al., 2016).

Although, it is present in small quantity, it can play a vital

role in metabolic reactions affected by abiotic stresses

(Luo et al., 2010). Furthermore, it is now widely reported

that trehalose can be readily taken up when applied

exogenously and plays a role in plant protection against

drought stress (Akram et al., 2016). For example,

exogenous application of trehalose at the range of 10-30

mM was found to be very effective in improving plant

biomass, antioxidative potential, nutrients status and

FIRDOS KOSAR ET AL., 956

chlorophyll pigments etc. in different plants e.g. maize

(Ali & Ashraf, 2011) and radish (Shafiq et al., 2015;

Akram et al., 2016) etc.

Sunflower (Helianthus annuus L.) being a high

yielding oil-seed crop, is ranked as the world’s fourth

largest oil-seed crop (Andaleeb et al., 2008; Akram et al.,

2012). Furthermore, it has a short life span (90-120 days)

and can be grown twice in a year without disturbing any

cash crop cultivation (Hussain et al., 2013). Drought

stress is reported to significantly reduce sunflower head

diameter, number of achenes along with their oil contents

and finally seed oil yield (Oad et al., 2002). Earlier, Sayed

& Gadallah (2002) reported that application of 5 and 10

mg L-1 thiamin either through root growing mediumor as

foliar spray on salt stressed sunflower plants enhanced dry

mass, chlorophyll contents, soluble sugar levels, and

absorption of K+. Similarly, Hussain et al., (2008)

externally applied 0.724 mM salicylic acid (SA) and 100

mMglycinebetaine which neutralized the adverse effects

of water deficiency on achene yield of sunflower whereas

the quality of oil contents remained unaltered. According

to another report, foliar spray of 20 and 80 mg L-1 of 5-

aminolevulinic acid proved to be effective in improving

growth in sunflower both in saline stress and non-stress

conditions (Akram & Ashraf, 2011). The principal

objective of the present study was to examine if foliar

application of trehalose could mitigate the adverse effects

of drought on plant biomass, different gas exchange

attributes, pattern of accumulation of some key non-

enzymatic metabolites such as proline and glycinebetaine

in two commercially grown sunflower cultivars.

Materials and Methods

Experimental site: This study on the role of exogenous

supply of trehalose in mitigating the effect of drought

stress on sunflower (Helianthus annuus L.) plants was

conducted in the Botanical Garden of the GCUF, Pakistan

from January to March in the spring season, 2015. The

seed (achenes) of two sunflower cultivars Hysun 33 and

FH 598 were collected from the Ayub Agricultural

Research Institute Faisalabad, Pakistan.

Plant growth and external applications: The crop was

planted in plastic pots (height 24.5 cm and diameter 21

cm). Pot soil (7 kg) was filled in each plastic pot. The

seeds were dipped in distilled water for two hours before

sowing. Ten seeds of each sunflower cultivar were sown

in each pot. Thinning was done after 15 days of planting

so as to maintain five plants in each pot.

Drought stress treatment: After three-week growth,

water stress treatments (100% and 60% of field capacity)

were applied. The moisture contents of drought hit pots

were monitored and maintained regularly considering the

weight of each pot equal to that calculated for 100 or 60%

field capacities through addition of normal irrigation, if

needed. At the vegetative stage, after 4 weeks of drought

stress treatment, four levels (0, 10, 20 and 30 mM) of

trehalose [formula, C12H22O11, molecular weight, 342.29

g/mol, density 1.58g/cm3, Company MP Bio USA and

100% pure] were applied as a foliar spray. The stock

solution of trehalose was prepared by dissolving 11.34

g/Ltrehalosein distilled water. Tween 20 (0.1%) was

added to varying concentrations of trehalose prior to foliar

spray. Before foliar spray, all pots were covered witha

paper to avoid addition of solution to the root growing

medium. All plants were allowed to grow for a further

two weeks and then the data for the following attributes

were determined.

Plant biomass: -Plant biomass (dry and fresh weights of

roots and shoots) of the sunflower plants was determined

individually after two weeks of spray. After recording the

data of fresh weights, the plants were placed in an oven at

60°C for 3 days and then dry weights measured.

Gas exchange characteristics: Infra Red Gas Analyzer

(IRGA) Ci-301 PS (CID, INC, USA), a portable

photosynthesis apparatus was used to measure gas exchange

parameters. Data recording time was 10.00 am to 11.00 am,

when sunflower leaves experienced irradiance just close to

1200 micromol/m2/s. Leaf temperature was 25 + 5°C.

Chlorophyll contents: Different steps for chlorophyll a

and b determination were followed as described by Arnon

(1949).

Leaf proline estimation: Proline estimation was

determined according to the protocol of Bates et al., (1973).

Glycinebetaine content (GB): The method described by

Grieve& Grattan (1983) was followed for the

measurement of glycinebetaine contents.

Statistical analysis: To test the significance level of each

treatment, analysis of variance (ANOVA) wasemployed

on the recorded data.

Results

Imposition of water-deficit stress (60% field

capacity) significantly (p≤0.001) suppressed the plant

biomass, i.e., shoot and root fresh and dry weights of both

Hysun 33 and FH 598 sunflower cultivars (Fig. 1). Foliar-

applied varying levels (10, 20 and 30 mM) of trehalose

significantly improved the shoot fresh weight (p≤0.001),

shoot dry weight (p≤0.01) and root dry weight (p≤0.01) of

both sunflower cultivars under stress and non-stress

conditions of both sunflower cultivars, cv. Hysun 33 was

relatively superior to cv. FH 598 in fresh and dry weights

of shoots and roots under water deficit conditions (Fig. 1).

A significant (p≤0.05) reduction in chlorophyll

apigment and a non-significant effect on chlorophyll

bwere observed due to water limited supply in both

sunflower cultivars. Exogenously applied trehalose was

proved effective in improving chlorophyll a and b

pigments in both sunflower cultivars. Of all levels of

trehalose used 20 and 30 mM were more effective in

improving chlorophyll pigments particularly chlorophyll

b contents in both sunflower cultivars. The response of

both sunflower cultivars was similar in accumulation of

chlorophyll a and b pigments under stress and non-stress

conditions (Fig. 1).

TREHALOSE-INDUCED DROUGHT TOLERANCE 957

Fig. 1. Shoot and root fresh and dry weights, chlorophyll a and b contents of water-stressed and non-stressed plants of two

cultivars of sunflower (Helianthus annuus) subjected to foliar-applied varying levels of trehalose (mean ± S.E.) D, Drought;

T, Trehalose; Cv, Cultivars; ns, Non-significant, *, ** and ***, significant at 0.05, 0.01 and 0.001 levels, respective.

FIRDOS KOSAR ET AL., 958

Fig. 2. Photosynthetic rate (A), transporation rate (E), stomatal conductance (gs) sub-stomatal CO2 concentration (Ci),

water use efficiency (A/E), Ci/Ca of two cultivars of sunflower (Helianthus annuus) subjected to foliar-applied varying

levels of trehalose (mean ± S.E.) D, Drought; T, Trehalose; Cv, Cultivars; ns, Non-significant, *, ** and ***, significant

at 0.05, 0.01 and 0.001 levels, respective.

TREHALOSE-INDUCED DROUGHT TOLERANCE 959

Fig. 3. Leaf proline and glycinebetaine contents of water-stressed and non-stressed plants of two cultivars of sunflower

(Helianthus annuus) subjected to foliar-applied varying levels of trehalose (mean ± S.E.) D, Drought; T, Trehalose; Cv,

Cultivars; ns, Non-significant, *, ** and ***, significant at 0.05, 0.01 and 0.001 levels, respective.

Data for different gas exchange characteristics showed

that drought stress (60% field capacity) significantly

suppressed the photosynthetic rate (A), transpiration rate

(E), stomatal conductance (gs), sub-stomatal CO2

concentration (Ci) and Ci/Ca ratio, but increased the water-

use-efficiency (WUE) of both sunflower cultivars.

Exogenous application of trehalose was effective in

improving all the earlier-mentioned gas exchange

characteristics in both sunflower cultivars under stress and

non-stress conditions. Of both sunflower cultivars, cv.

Hysun 33 was superior to the other cultivar in A, E, Ci,

Ci/Ca, while the reverse was true in WUE. The response of

both sunflower cultivars to gs was similar under control and

moisture deficit conditions (Fig. 2).

Under water deficit conditions, leaf proline and

glycinebetaine (GB) accumulations increased considerably

(p≤0.001) in both sunflower cultivars (Fig. 3). Trehalose-

treated plantsof both sunflower cultivars were significantly

(p≤0.01; 0.001) higher in proline and GB accumulation,

particularly under water stress conditions. Both sunflower

cultivars were similar in response to proline and GB

accumulation under water stressed conditions (Fig. 3).

Discussion

During different phases of life, plants are naïve to be

exposed to various stresses like water shortage, soil salinity,

extremes of temperature etc. To survive under such adverse

cues, plants employ different defensive adaptive

mechanisms. However, the most promising is

osmoregulation (Holmstrom et al., 2000). During this

activity, certain low molecular weight carbohydrates,

proteins and amino acids play an important role (Hasegawa

et al., 2000; Akram et al., 2016). If the levels of these low

molecular weight substances are low within the plant body,

the plant is generally not able to withstand harsh

environmental cues. Thus alternatively as a shot-gun

approach, such substances can be applied exogenous to

bring their intrinsic concentrations to the optimum levels.

Foliar spray of such organic compounds has been

advocated as a viable strategy to impart vitality to plants so

that they can thrive well under unfavorable stress

conditions. Of various sugars involved in stress tolerance,

trehalose is known to stabilize the desiccated enzymes,

proteins and lipids effectively as well as it protects

biological membranes from damage during dehydration

FIRDOS KOSAR ET AL., 960

(Garg et al., 2002). In the present investigation, water

deficit conditions significantly impaired growth parameters

(shoot and root fresh and dry weight) of both sunflower

cultivars (Hysun 33 and FH 598). However, foliar

application (10, 20 and 30 mM) of trehalose significantly

improved these growth parameters (Fig. 1). These results

can be related to earlier studies which also used exogenous

application of trehalose on different plants under stressful

conditions as in maize (Zeid, 2009), Catharanthus roseus

(Chang et al., 2014), Brassica species (Alam et al., 2014),

wheat (Ibrahim & Abdellatif, 2016), and radish (Akram et

al., 2016), etc. The trehalose-induced plant growth

improvement was suggested to be associated with

upregulation of oxidative defense system, maintenance of

turgor potential and nutritional balance as well as

suppression in the levels of reactive oxidants under stress

conditions (Alam et al., 2014; Akram et al., 2016).

Upon exposure to abiotic stresses, reactive oxygen

species (ROS) are generated in plants which rapidly

deteriorate the chlorophyll contents (Baker et al., 2007).

Similar results were observed in the present study. The

moisture deficient stress significantly reduced the

chlorophyll contents, particularly chlorophyll a contents.

The trehalose treatment significantly improved both

photosynthetic pigments in both sunflower cultivars under

stress and non-stress conditions (Fig. 1). Similar findings

were reported under water stress in Vicia faba (Gadallah,

1999), wheat (Kamran et al., 2009), and maize (Ashraf et

al., 2007). Likewise, Akram et al., (2016) reported that in

radish plant, trehalose pretreatment caused a significant

increase in chlorophyll a contents. In Fenugreek plant,

Sadak (2016) reported enhanced levels of photosynthetic

pigments in trehalose-treated plants as compared to those

in the non-treated plants.

Stomatal closure is contemplated as the first line of

defense against the onset of soil desiccated conditions. This

modification in stomatal regulation affects photosynthetic

activity due to denaturation of proteins related to

photosystems. Data recorded for different gas exchange

components in this experiment depicted that limited soil

moisture conditions negatively affected the photosynthetic

rate (A), transpiration rate (E), stomatal conductance (gs),

sub-stomatal CO2 concentration (Ci) and Ci/Ca ratio, but

they increased the water-use-efficiency (WUE) of both

sunflower cultivars. The foliar spray of trehalose played a

beneficial role in all the above-mentioned gas exchange

attributes in both sunflower cultivars but an opposite trend

was noted for water use efficiency. In this respect, Ali&

Ashraf (2011) found that treatment of trehalose

significantly improved the A, E, Ci, gs, A/E, and Ci/Ca in

maize cultivars under drought stress and non-stress

conditions. Similarly, Akram et al., (2016) reported that

priming radish seed with trehalose significantly raised plant

water use efficiency. This discussion affirms that trehalose

spray on plants induces positive adjustment in sunflower

plants between carbon utilization and transpiration.

The compatible osmoprotectants such as

glycinebetaine and proline play a significant role in plants

to endure the prevailing abiotic stress conditions. These

solutes upregulate plant key physiological processes

under stress adversaries and make them acclimatized

(Nounjan et al., 2012). In the present study, leaf proline

and glycinebetaine (GB) accumulation increased

considerably in both sunflower cultivarsunder water

deficit stress. The trehalose-treated plants of both

sunflower cultivars were significantly higher in proline

and GB accumulation, particularly under water stress

conditions (Fig. 3). Our results are analogous to those of

Alam et al., (2014) who observed enhanced accumulation

of proline and GB in Brassica campestris seedlings

during a combined treatment of trehaloseand drought

stress. Not only foliar spray, but also pre-sowing seed

treatment with trehalose induced higher levels of proline

in water stressed radish plants (Akram et al., 2016) but in

contrast, Nounjan et al., (2012) reported that in salt

stressed rice seedlings, externally applied (rooting

medium) trehalose suppressed endogenous proline levels.

In the case of GB accumulation in drought stressed

sunflower plants, our results are in agreement with those

of some earlier research reports e.g., sunflower (Akram et

al., 2012), and radish (Shafiq et al., 2015) under water

shortage conditions. They suggested that trehalose

induced increase in GB accumulation could play an

important role in drought stress tolerance. In conclusion, exogenous application of trehalose

significantly improved the plant growth in terms of shoot fresh and dry weights, and root dry weight of both sunflower cultivars under water deficit stress. In addition, trehalose-induced significant improvement was also observed in chlorophyll a and b, A, E, gs, Ci, Ci/Ca, WUE, proline and GB contents predominantly under water stress conditions. Of all levels, 20 and 30 mM of trehalose were more effective for improving growth, gas exchange characteristics and osmoprotection components in both sunflower cultivars.So, it can be suggested that foliar applied trehaloseis an effective mode for achieving enhanced drought stress tolerance in sunflower plants by improving osmoprotection and gas exchange characteristics.

Acknowledgements

This research work is a part of Ph.D. thesis of Miss

Firdos Kosar.

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(Received for publication 18 August 2017)